The present invention relates to immunoassays for non-collagen cartilage proteins and their fragments in biological samples such as body fluids. Such proteins and protein fragments may serve as an index of joint disease.
A number of diseases are associated with increased turnover of the extra cellular matrix of tissues or organs in the mammalian body. As examples of such diseases or pathological conditions, arthritic diseases involves increased turnover of tissues of the joint, osteoporosis is associated with increased turnover of bone tissue, psoriasis and scleroderma is associated with increased turnover of skin and various cancer types are also associated with increased tissue turnover of the disease affected tissue(s) or organ(s). Of special relevance in these conditions is a quantification of the catabolic processes associated with the disease as these often predominates and results in degradation of the affected tissues or organs. Such catabolic process can be difficult to quantify, and specific biochemical markers for monitoring such processes would be of great clinical utility for diagnosis, monitoring and assessing prognosis of the diseases. Furthermore, such markers could be used in pre-clinical and clinical research for identification and assessment of new therapeutic agents and for optimising treatment dose and treatment modality. However, even though a need for such markers clearly exists it has been difficult to identify and develop such biochemical markers and very few such markers exist today for routine use in clinical management of disease or research into disease mechanisms and development of new therapeutic agents.
The present invention provides a general method for identification of biochemical markers specific for catabolic processes in a mammalian tissue or organ. This method has been applied for identification of new markers of cartilage turnover; a process which is of relevance for all arthritic diseases, as described below.
Increased awareness of the early biochemical and structural changes in cartilage-related diseases in combination with the introduction of new disease suppressive agents has created the need to develop improved tools to assess disease severity and prognosis. Thus the need for sensitive simple and reliable markers for cartilage degradation is evident, and such markers will fulfill important clinical purpose for management of arthritic disease.
As of today, no tissue specific biochemical marker exists which is of general use for diagnosis and monitoring of cartilage degradation in Rheumatoid Arthritis (RA) and Osteoarthritis (OA) patients. Markers such as C-reactive protein (CRP), rheumatoid factor (RF) and erythrocyte sedimentation rate (ESR) are frequently applied in RA. These markers provide information about the inflammatory reactions in the disease, but are not specific for joint diseases, and in spite of their wide-spread use, their clinical utility is still discussed. At present one of the best ways to obtain information about the status of the (individual) joints in arthritis patients is radiological examination.
Measurement of metabolites, such as hyaluronates and aggrecan fragments arising from destruction of the specific organ (the joints) affected by the arthritis has been reported, for review please see Wollheim (Wollheim, 1996). The two main arthritic diseases, RA and OA are briefly described below.
Osteoarthritis:
Osteoarthritis (OA) is a chronic disease characterized by degenerative changes of the joints and bone with destruction of the cartilage and reactive formation of bone in the periphery of the joint. OA is a common disease, with an increasing incidence with age. At the age of 50 approximately 20% have symptoms of OA and radiologically almost 90% exhibit OA-related changes.
One of the earliest events that can be detected histologically is the loss of proteoglycans (aggrecan) from the tissue (Flannerly et al 1992). The likely mechanism is the fragmentation of aggrecan by proteinases. The generation of aggrecan fragments is detrimental to the proper functioning of cartilage. In later stages of the disease also collagen fibrils are degraded (Eyre et al 1991) and the surface of the cartilage is eroded due to loss of tissue (Inerot & Heineg{dot over (a)}rd, 1982).
Rheumatoid Arthritis:
Rheumatoid arthritis (RA) is an autoimmune disease, where the patients own immune system attacks the joints, causing inflammation and subsequent degradation of the cartilage matrix. The disease affects approximately 1% of the population. At present the underlying causes responsible for initiation of the disease are unknown, however studies of its epidemiology show that genetic as well as environmental factors contribute to the pathogenesis.
RA is characterized by chronic inflammation associated with erosion of both cartilage and bone. In advanced late stages of the disease, radiographs show characteristic evidence of cartilage loss and bone erosion at the joint margin. However much damage may be done early in the disease, before radiographs change. Sensitive indices of the current cartilage and bone changes in RA would therefore be of potential value to clinicians.
The present invention describes methods for identifying such markers and also methods for developing and applying assays for such markers for management of arthritic disease as well as for developing new therapeutic or disease management methods for arthritic disease.
One aim of the present invention is to provide new methods and procedures for identifying potential markers of cartilage degradation based on identification of isomerised and/or optically inverted fragments of cartilage-derived proteins. Such markers will provide a significant advantage for the clinical management of OA, RA as well as other diseases affecting joint metabolism. Furthermore the use of such markers for management of arthritic disease and development of anti-arthritic therapeutic agents is described.
The Extracellular Matrix of Cartilage:
Cartilage matrix is synthesized, degraded, organized and maintained by a sparse population of chondrocytes (making up 0.5-2% of the total cartilage volume) (Poole et al 1994, Poole & Dieppe 1994). These cells are protected from the potentially damaging forces of mechanical function by the extra-cellular matrix (ECM) they produce.
The properties of cartilage are critically dependent upon the structure and integrity of the ECM. Thus, normal turnover is a conservative process in which the rate of matrix degradation does not exceed the rate at which it is replaced. There is a slow constant turnover of the ECM with chondrocytes both degrading and synthesizing matrix proteins. Healing of cartilage is dependent entirely on surviving chondrocytes near the margins of the injury. In adults these cells mediate essentially no repair.
A number of cartilage proteins have been identified and characterized. The main structural component of cartilage is made up of collagen fibers, predominantly composed of collagen type II with minor amounts (typically less than 10%) of collagen type IX and type XI. Interspaced between the collagen network are long chains of the negatively charged polysaccharide hyaluronic acids, to which several large proteoglycans are attached. The major proteoglycan is aggrecan. Aggrecan is a large protein with a molecular mass of 2600 kDa with a core protein of 220 kDa (Doege et al 1991). The protein is composed of 93% glycosaminoglycans (about 87% chondroitin sulphate and 6% keratan sulphate) and 7% protein. Aggrecan is a major component of cartilage where it accounts for about 10% of the dry weight of cartilage. The oligosaccharide moieties of aggrecan are highly negatively charged which draws water into the tissue as it creates a large osmotic swelling pressure. The water thus swells and expands the aggrecan-rich matrix. This places the collagen network under tension and an equilibrium is achieved when tension in the collagen network balances the swelling pressure (i.e., when no more water enters the tissue because the force is insufficient to stretch the collagen network any further). The aggrecan molecule is made up of 7 different domains. At the N-terminus there are two structurally related globular domains termed G1 and G2, separated by a short region known as the interglobular domain (IGD). Although the function of the G2 domain has not been determined the G1 domain is known to serve as a ‘linker’ to hyaluronan and is responsible for the formation of large aggrecan aggregates (Hardingham & Muir, 1972). The G1 domain is also in contact with cartilage link protein (see below). C-terminally to the G2 domain there is a long region consisting of two glycosaminoglycan-rich regions. The first is a domain rich in keratan sulphate (KS), whereas the other is composed of two domains rich in chondroitin sulphate (CS). At the C-terminus aggrecan possesses a third globular domain G3. The G3 domain seems to be lost soon after synthesis and secretion, the consequence of loss of the G3 domain is not yet understood (Hardingham & Fosang, 1992). Current evidence indicates that aggrecan fragments released during degradation of the cartilage matrix are heterogeneous products and may reflect various stages of disease progression depending on which fragments are monitored. In early or non-cartilage destructive diseases no or only small amounts of fragments related to the G1 domain are released (Saxne & Heineg{dot over (a)}rd 1992). Increased release of G1 related fragments, only accompanies the more profound and presumably irreversible cartilage derangement seen in patients with more damaged joints (Saxne & Heineg{dot over (a)}rd 1995).
A number of non-collagen cartilage proteins have been described although not all of these proteins are specific in cartilage, they play important structural and/or functional roles in the tissue, and may potentially serve as markers of cartilage turnover.
Cartilage intermediate layer protein (CILP) is noncollagenous cartilage protein composed of a single polypeptide chain with a molecular weight of 91.5 kDa, including N-linked oligosaccharides (Lorenzo et al. 1998a and 1998b). The protein is synthesized by chondrocytes and located to the interterritorial cartilage. It is neither found in the superficial nor deepest regions of the articular cartilage. CILP has been reported to increase with age and has been suggested to be a marker of early OA.
Cartilage Oligomeric Matrix Protein (COMP) is a non-collagenous extra-cellular matrix protein found predominantly in cartilage, but also in tendon, ligament and meniscus (Muller et al 1998). Several mesenchymal cells including synoviocytes and dermal fibroblasts produce substantial amounts of COMP (Dodge et al 1998). The physiological function of the protein is not known. COMP is a large disulfide-linked pentameric protein with a molecular weight of each monomer of about 100 kDa. The protein belongs to the thrombospondin family and it displays a high homology to thrombospondin 1-4 (Adams & Tucker 2000). In vitro data indicate that COMP may mediate cell binding within the cartilage matrix in accordance with the function of other members of the thrombospondin family. Furthermore, it is likely that COMP participates in regulation of collagen fibril formation (Rosenberg et al 1998). The major source of COMP within the joint appears to be fibroblastic cells in sub-synovial tissue. In cartilage COMP may be found as both the intact pentamer and in various fragments (43-160 kDa) generated by the action of MMP's as well as other proteases (Saxne & Heineg{dot over (a)}rd 1992, Ganu et al 1998). In OA cartilage the proportion of degraded COMP is higher than in normal cartilage even though the total amounts of COMP appears to be similar (Vilim et al 1997, Niedhart et al 1997). In RA cartilage there is a net loss of COMP, and the COMP fragments appear to be of smaller size than those seen in OA (Clarke et al 1999, Vingsbo-Lundberg et al 1998).
A number of COMP assays based on both polyclonal and monoclonal antibodies have been described in the literature, but the exact epitope specificity of the assays is generally not characterized (Saxne & Heineg{dot over (a)}rd 1992, Niedhart et al 1997). A number of cross-sectional studies have demonstrated elevated COMP levels in patients with OA, RA and other diseases affecting joint metabolism (Sharif et al 1995, Vingsbo-Lundberg et al 1998, Clarke et al 1999, Niedhart et al 1997).
Perlecan is a heparin sulphate proteoglycan that is expressed in all basement membranes, cartilage and several other mesenchymal tissues during development. Perlecan binds growth factors and interacts with various extracellular matrix proteins and cell adhesion molecules. Relatively high levels of perlecan have been found in mature cartilage, and in vitro experiments have suggested that perlecan supports chondrocyte differentiation (Costell et al 1999).
Biglycan and decorin belong to the family of small leucine rich proteoglycans. They are relatively highly expressed in foetal cartilage, but are found in lower amounts in adult cartilage. Their functional role in cartilage is not fully elucidated but they may be involved in maintaining the structural integrity of the collagen fiber network and the hyaluronic acid mesh (Knudson & Knudson 2001). Furthermore they may play a role in chondrogenesis and cartilage degradation.
Fibrillin-1 (Fib-1) is a connective tissue protein, with an estimated molecular mass of 350,000 D. Fibrillin 1 is found in extracellular microfibrils in a variety of connective tissues. The protein has a widespread distribution in the connective tissue matrices of skin, lung, kidney, vasculature, cartilage, tendon, muscle, cornea, and ciliary zonule (Sakai et al 1986). The functional relationships between this glycoprotein and other components of the microfibrils and elastic fibers are unknown. Synthesis of fibrillin-1 correlates with late morphogenesis and the appearance of well-defined organ structures (Zhang and Ramirez 1995). It is widely expressed in developing limbs and digits, especially in the perichondrium (Keene et al 1997). Fib-1 appears as a loose meshwork of fibers within cartilage matrix by 20 weeks of fetal gestation. Until early adolescence, Fib-1 forms loose bundles of microfibrils within cartilage (Keene et al; 1997). However, by late adolescence, broad banded fibers composed of Fib-1 are found accumulated pericellularly within cartilage probably as laterally packed and crosslinked microfibrils. It has been proposed that fibrillin-1 provides force-bearing structural support to extracellular microfibrils and that it may growth-regulating functions in the perichondrium (Keene et al. 1997, Zhang and Ramirez 1995).
Protocadherins constitute a large family belonging to the cadherin superfamily and function in different tissues of a wide variety of multicellular organisms. Protocadherins have unique features that are not found in classic cadherins. Little is known about the Protocadherin subfamily gamma.
In joint diseases, the rate of degradation of matrix often exceeds the rate of synthesis, and, as consequence, the tissue becomes thin and mechanically weak (Heineg{dot over (a)}rd et al 1999). As the catabolic processes dominate in the arthritic disease states, fragments of cartilage proteins is produced, and such fragments can be measured in synovial fluid, serum and urine as markers of the cartilage degradation.
Joint diseases and related conditions involving abnormalities in the metabolism of cartilage cause widespread disability. Erosion of the articular cartilage is a typical finding in degenerative and inflammatory joint diseases, such as OA and RA.